Chemical enhancement of CRISPR/Cas9 mediated site-specific genome engineering. Abstract Programmable nucleases, including Zinc Finger Nucleases, TALENs, meganucleases and the CRISPR/Cas9 system allow for site-specific genome engineering. The ability to make targeted genetic modifications has opened up a wide variety of options for scientists in industry and academia and in both therapeutic and biotechnology disciplines. During precision genome engineering, a site specific DSB is generated through nuclease activity. The DSB is repaired by the error prone non-homologous end joining (NHEJ) pathway or by homology directed repair (HDR). Genetic recombination in mammalian systems through the HDR pathway is an extremely inefficient process and cumbersome laboratory methods are required to identify the accurate, desired events. This is further compromised by the activity of the competing DNA repair pathway, NHEJ, which repair the majority of DNA DSBs and can often lead to insertion and deletions resulting in mutagentic events. Recent studies have shown that decreasing NHEJ activity in vivo results in an increase in HDR activity, and this phenomena can be exploited to increase the efficiency of HDR mediated CRISPR/Cas9 precision genome engineering. We have developed a series of small molecule chemical inhibitors that inhibit the DNA binding activity of Ku, a protein necessary for initiation of the NHEJ pathway. Preliminary in vitro and cellular data has shown that Ku DNA binding activity is abolished in the presence of the inhibitors in a potent and specific fashion. I a single aim we will address the ability of these inhibitors to decrease NHEJ and subsequently increase HDR mediated genome engineering using the CRISPR/Cas9 system. Completion of these studies will allow us to move forward with a commercialization plan to market the inhibitors to parties interested in increasing the efficiency and specificity of CRISPR/Cas9 genome engineering.